Summary Synucleinopathies are a group of neurodegenerative disorders that have been associated to the misfolded amyloid protein ?-synuclein in brain. The misfolded ?-synuclein can aggregate into polymorphic fibrils, displaying distinct biological activities and contributing differently to the diseases. However, the underlying mechanisms remain unclear. To address this, we will integrate both structural and functional approaches to study recombinant wild type, disease-related mutants, and brain-derived ?-synuclein fibrils. First, we will use cryo-Electron Microscopy (cryo-EM), combined with other structural methods, to achieve near-atomic structures of recombinant full-length fibrils of wild type and disease mutant ?-synuclein (Aims 1-2). We will then go beyond structure determination and utilize cellular assays of seeding and toxicity to explore the biological activities of both wild type and disease mutant ?-synuclein fibrils. By comparing the structures of these fibrils, we will determine structure elements responsible for their biological activity, and correlate structural differences to seeding and toxicity for a better understanding of their structure-activity relationship (Aim 2). Finally, we will determine cryo-EM structures of in vivo ?-synuclein fibrils derived from the patient brains of synucleinopathies. Thus we can assess the pathological relevance of the determined recombinant fibril structures and relate structural features observed in ?-synuclein fibrils to different disease states (Aim 3). Our integrated approach, connecting structure and biological activity of ?-synuclein fibrils of different forms or sources, will reveal atomic understanding of the underlying mechanisms and provide therapeutic targets suitable for future drug development that precisely targets ?-synuclein aggregation to stop synucleinopathies.